OBJECTIVES: a) The overall objective of this entire research project is to purify sufficient quantities of D-lactic dehydrogenase from two strains of Moraxella (ATCC19965, 19975) from one strain of Neisseria (ATCC8193) and anthranilate synthetase from Moraxella (ATCC19965) for the preparation of rabbit antibody specific against these enzymes. The antisera will then be used to establish the degree of antigenic similarity for these enzymes from these and other catalogued strains of Moraxella and Neisseria. The potential use of these antisera for routine determinative purposes will then be assessed by testing them against clinical isolates identified as various species of Moraxella, Neisseria, Acinetobacter, or Mima-Herellea. b) The goal for the second year of this project was to devise a purification procedure, using Moraxella osloensis (ATCC19965) as a source of D-lactic dehydrogenase (D-LDH), which could also be used for the purification of D-LDH from Moraxells non-liquefaciens (ATCC19975) and Neisseria (ATCC8193) and to scale up the procedure in order to obtain amounts (2-3 milligrams) sufficient for antiserum production in rabbits. A procedure has been devised for isolating cytoplasmic membranes from the organisms (ATCC19965) using lysozyme (750e.u.) in a Tris buffer (10 to the minus 2nd power M, pH 8) containing EDTA (10 to the minus 4th power M) and sucrose (20%). Washing the membranes with buffer removed cytoplasmic proteins as well as some envelope proteins. The membrane-bound D-LDH was recovered following solubilization of the membrane with 50 mM phosphate buffer (pH 7.5) containing Triton-X-100 (1%) and EDTA (10 micromolar). Enrichment of D-LDH is approximately 7 fold using this procedure compared to the specific activity of the enzyme observed in cell free preparations obtained using ultrasonic disruption techniques. A greater enrichment of initial D-LDH activity has been achieved by replacing EDTA with 10 micromolar MgCl2 in the membrane-solubilization buffer. Chromatography of the enzyme of DEAE cellulose and affinity chromatography have yielded preparations estimated to be approximately 70% pure as judged by polyacrylamide gel electrophoresis (PAGE). Both protein and activity stains have been used to assess enzyme purit (Text Truncated - Exceeds Capacity)